Human Salivary Gland Tumors Research Articles

Effects of cellular radioresponse on therapeutic helium-, carbon-, oxygen-, and neon-ion beams: a simulation study

Objective. Helium, oxygen, and neon ions in addition to carbon ions will be used for hypofractionated multi-ion therapy to maximize the therapeutic effectiveness of charged-particle therapy. To use new ions in cancer treatments based on the dose-fractionation protocols established in carbon-ion therapy, this study examined the cell-line-specific radioresponse to therapeutic helium-, oxygen-, and neon-ion beams within wide dose ranges. Approach. Response of cells to ions was described by the stochastic microdosimetric kinetic model. First, simulations were made for the irradiation of one-field spread-out Bragg peak beams in water with helium, carbon, oxygen, and neon ions to achieve uniform survival fractions at 37%, 10%, and 1% for human salivary gland tumor (HSG) cells, the reference cell line for the Japanese relative biological effectiveness weighted dose system, within the target region defined at depths from 90 to 150 mm. The HSG cells were then replaced by other cell lines with different radioresponses to evaluate differences in the biological dose distributions of each ion beam with respect to those of carbon-ion beams. Main results. For oxygen- and neon-ion beams, the biological dose distributions within the target region were almost equivalent to those of carbon-ion beams, differing by less than 5% in most cases. In contrast, for helium-ion beams, the biological dose distributions within the target region were largely different from those of carbon-ion beams, more than 10% in several cases. Significance. From the standpoint of tumor control evaluated by the clonogenic cell survival, this study suggests that the dose-fractionation protocols established in carbon-ion therapy could be reasonably applied to oxygen- and neon-ion beams while some modifications in dose prescription would be needed when the protocols are applied to helium-ion beams. This study bridges the gap between carbon-ion therapy and hypofractionated multi-ion therapy.

A methodology to abridge microdosimetric distributions without a significant loss of the spectral information needed for the RBE computation in carbon ion therapy.

In order to compute the relative biological effectiveness (RBE) of ion radiation therapy with the Mayo Clinic Florida microdosimetric kinetic model (MCF MKM), it is necessary to process entire microdosimetric distributions. Therefore, a posteriori RBE recalculations (i.e., for a different cell line or another biological endpoint) would require whole spectral information. It is currently not practical to compute and store all this data for each clinical voxel. To develop a methodology that allows to store a limited amount of physical information without losing accuracy in the RBE calculations nor the possibility of a posteriori RBE recalculations. Computer simulations for four monoenergetic 12 C ion beams and a 12 C ion spread-out Bragg peak (SOBP) were performed to assess lineal energy distributions as a function of the depth within a water phantom. These distributions were used in combination with the MCF MKM to compute the in vitro clonogenic survival RBE for human salivary gland tumor cells (HSG cell line) and human skin fibroblasts (NB1RGB cell line). The RBE values were also calculated with a new abridged microdosimetric distribution methodology (AMDM) and compared with the reference RBE calculations using the entire distributions. The maximum relative deviation between the RBE values computed using the entire distributions and the AMDM was 0.61% (monoenergetic beams) and 0.49% (SOBP) for the HSG cell line, while 0.45% (monoenergetic beams) and 0.26% (SOBP) for the NB1RGB cell line. The excellent agreement between the RBE values computed using the entire lineal energy distributions and the AMDM represents a milestone for the clinical implementation of the MCF MKM.

The Mayo Clinic Florida microdosimetric kinetic model of clonogenic survival: formalism and first benchmark against in vitro and in silico data

Objective. To develop a new model (Mayo Clinic Florida microdosimetric kinetic model, MCF MKM) capable of accurately describing the in vitro clonogenic survival at low and high linear energy transfer (LET) using single-event microdosimetric spectra in a single target. Methodology. The MCF MKM is based on the ‘post-processing average’ implementation of the non-Poisson microdosimetric kinetic model and includes a novel expression to compute the particle-specific quadratic-dependence of the cell survival with respect to dose (β of the linear-quadratic model). A new methodology to a priori calculate the mean radius of the MCF MKM subnuclear domains is also introduced. Lineal energy spectra were simulated with the Particle and Heavy Ion Transport code System (PHITS) for 1H, 4He, 12C, 20Ne, 40Ar, 56Fe, and 132Xe ions and used in combination with the MCF MKM to calculate the ion-specific LET-dependence of the relative biological effectiveness (RBE) for Chinese hamster lung fibroblasts (V79 cell line) and human salivary gland tumor cells (HSG cell line). The results were compared with in vitro data from the Particle Irradiation Data Ensemble (PIDE) and in silico results of different models. The possibility of performing experiment-specific predictions to explain the scatter in the in vitro RBE data was also investigated. Finally, a sensitivity analysis on the model parameters is also included. Main results. The RBE values predicted with the MCF MKM were found to be in good agreement with the in vitro data for all tested conditions. Though all MCF MKM model parameters were determined a priori, the accuracy of the MCF MKM was found to be comparable or superior to that of other models. The model parameters determined a priori were in good agreement with the ones obtained by fitting all available in vitro data. Significance. The MCF MKM will be considered for implementation in cancer radiotherapy treatment planning with accelerated ions.

Esculetin has therapeutic potential via the proapoptotic signaling pathway in A253 human submandibular salivary gland tumor cells.

Esculetin is a natural lactone that is commonly derived from coumarins. According to previous experiments using human cancer cells, esculetin has potent antitumor activity; it also inhibits proliferation and induces the apoptosis of cancer cells. In the present study, the anti-proliferative effect of esculetin on the submandibular salivary gland tumor cell line, A253, was evaluated via in vitro and in vivo analyses. Furthermore, the anti-cancer effects of esculetin in A253 cells and a xenograft model of salivary gland tumors were determined using 3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bromide and TUNEL assay, apoptosis protein array, quantitative polymerase chain reaction and western blot analysis. Esculetin (50-150 µM) was demonstrated to have an anti-proliferative effect in the A253 cell line in vitro; this observed effect was dependent on the dose and duration of treatment. Esculetin also increased the levels of Bax, cleaved caspase-3, cleaved-9 and cleaved poly (ADP-ribose) polymerase apoptosis-related proteins, and decreased the expression levels of the Bcl-2 anti-apoptotic protein. With respect to apoptosis regulation, esculetin significantly decreased the proliferation of tumor cells in a xenograft model (100 mg/kg/day) for 18 days. Overall, esculetin could be a potential oral anticancer drug against salivary gland cancer.

Cytotoxicity of Propolis Extracts obtained using Dichloromethane and Hexane Solvent on Human Salivary Gland Tumor Cell Line.

This in vitro study aimed to investigate the effect of propolis extracts from two different solvents on human submandibular salivary gland (HSG) tumor cell line. Propolis was extracted by dichloromethane (DCM) and hexane (HEX). Crude extracts were prepared from 6.25 to 200 µg/mL in Dulbecco's modified eagle medium without serum. Flavonoid and total phenolic contents of crude extracts were measured using a modified colorimetric method. The cytotoxicity was evaluated by 3-[4, 5-dimethylthiazol-2-yl]-2,5 diphenyl-tetrazolium (MTT) assay and lactate dehydrogenase (LDH) release assay. The statistics were analyzed by independent sample t-test. Propolis extracts obtained using DCM and HEX exhibited comparable % yield (38.58 and 38.25) and physical characteristics and different amounts of flavonoid (0.439 ± 0.02 and 0.250 ± 0.01 mg catechin/g sample) and total phenolic compounds (3.759 ± 0.03 and 1.618 ± 0.03 mg gallic acid equivalents/g sample). The DCM group at 25, 50, 100, and 200 µg/mL as well as the HEX group at 50, 100, and 200 µg/mL significantly displayed a decrease in % cell viability and an increase in % cytotoxicity, compared with the untreated control group (P < 0.05). The DCM group showed the half-maximal inhibitory concentration (IC50) of MTT (42.93 ± 2.70) and LDH (34.94 ± 0.22). The HEX group showed the IC50 of MTT (61.30 ± 5.39) and LDH (42.32 ± 1.00). Propolis extracts obtained using both DCM and HEX are effective to inhibit HSG viability. Regarding to the cell morphological observation, MTT and LDH assays, propolis extracts obtained using DCM and HEX exhibited the cytotoxic effect on HSG tumor cell line. Based on our knowledge, this research demonstrates the first preliminary result suggesting propolis as a natural product of choice for salivary gland cancer prevention and therapy.

Adaptation of stochastic microdosimetric kinetic model to hypoxia for hypo-fractionated multi-ion therapy treatment planning

For hypo-fractionated multi-ion therapy (HFMIT), the stochastic microdosimetric kinetic (SMK) model had been developed to estimate the biological effectiveness of radiation beams with wide linear energy transfer (LET) and dose ranges. The HFMIT will be applied to radioresistant tumors with oxygen-deficient regions. The response of cells to radiation is strongly dependent on the oxygen condition in addition to radiation type, LET and absorbed dose. This study presents an adaptation of the SMK model to account for oxygen-pressure dependent cell responses, and develops the oxygen-effect-incorporated stochastic microdosimetric kinetic (OSMK) model. In the model, following assumptions were made: the numbers of radiation-induced sublethal lesions (double-strand breaks) are reduced due to lack of oxygen, and the numbers of oxygen-mediated lesions are reduced for radiation with high LET. The model parameters were determined by fitting survival data under aerobic and anoxic conditions for human salivary gland tumor cells and V79 cells exposed to helium-, carbon-, and neon-ion beams over the LET range of 18.5–654.0 keV μm−1. The OSMK model provided good agreement with the experimental survival data of the cells with determination coefficients >0.9. In terms of oxygen enhancement ratio, the OSMK model reproduced the experimental data behavior, including slight dependence on particle type at the same LET. The OSMK model was then implemented into the in-house treatment planning software for the HFMIT to validate its applicability in clinical practice. A treatment plan with helium- and neon-ion beams was made for a pancreatic cancer case assuming an oxygen-deficient region within the tumor. The biological optimization based on the OSMK model preferentially placed the neon-ion beam to the hypoxic region, while it placed both helium- and neon-ion beams to the surrounding normoxic region. The OSMK model offered the accuracy and usability required for hypoxia-based biological optimization in HFMIT treatment planning.

Microdosimetric measurements as a tool to assess potential in-field and out-of-field toxicity regions in proton therapy

Relative biological effectiveness (RBE) variations are thought to be one of the primary causes of unexpected normal-tissue toxicities during tumor treatments with charged particles. Unlike carbon therapy, where treatment planning is optimized on the basis of the RBE-weighted dose, a constant RBE value of 1.1 is currently used in proton therapy. Assuming a uniform value can lead to under- or over-dosage, not just to the tumor but also to surrounding normal tissue. RBE changes have been linked with dose/fraction, the biological endpoint and beam properties. Understanding radiation quality and the associated RBE can improve the prediction of normal-tissue toxicities.In this study, we exploited microdosimetry for characterizing radiation quality in proton therapy in-field, and off-beam at 20 (beam edge), 50 (close out-of-field) and 100 (far out-of-field) mm from the beam center. We measured the lineal energy y spectra in a water phantom irradiated with 152 MeV protons, from which beam quality as well as the physical dose could be obtained. Taking advantage of the linear quadratic model and a modified version of the microdosimetric kinetic model, the microdosimetric data were combined with radiobiological parameters (α and β) of human salivary gland tumor cells for assessing cell survival RBE and RBE-weighted dose.The results indicate that if a dose of 60 Gy is delivered to the peak, the beam edge receives up to 6 Gy while the close and far out-of-field regions receive doses on the order of 10−3 Gy and 10−4 Gy, respectively. The RBE estimate in-beam shows large variations, ranging from 1.0 ± 0.2 at the entrance channel to 2.51 ± 0.15 at the tail. The beam edge follows a similar trend but the RBE calculated at the Bragg peak depth is 2.27 ± 0.17, i.e. twice the RBE in-beam (1.05 ± 0.15). Out-of-field, the estimated RBE is always significantly higher than 1.1 and increases with increasing lateral distance, reaching the overall highest value of 3.4 ± 0.3 at a depth of 206 mm and a lateral distance of 10 mm. The combination of RBE and dose into the biological dose points to the beam edge and the end-of-range in-beam as the areas with the highest risk of potential toxicities.

Dose compensation based on biological effectiveness due to interruption time for photon radiation therapy.

To evaluate the biological effectiveness of dose associated with interruption time; and propose the dose compensation method based on biological effectiveness when an interruption occurs during photon radiation therapy. The lineal energy distribution for human salivary gland tumor was calculated by Monte Carlo simulation using a photon beam. The biological dose (Dbio) was estimated using the microdosimetric kinetic model. The dose compensating factor with the physical dose for the difference of the Dbio with and without interruption (Δ) was derived. The interruption time (τ) was varied to 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 75, and 120 min. The dose per fraction and dose rate varied from 2 to 8 Gy and 0.1 to 24 Gy/min, respectively. The maximum Δ with 1 Gy/min occurred when the interruption occurred at half the dose. The Δ with 1 Gy/min at half of the dose was over 3% for τ >= 20 min for 2 Gy, τ = 10 min for 5 Gy, and τ = 10 min for 8 Gy. The maximum difference of the Δ due to the dose rate was within 3% for 2 and 5 Gy, and achieving values of 4.0% for 8 Gy. The dose compensating factor was larger with a high dose per fraction and high-dose rate beams. A loss of biological effectiveness occurs due to interruption. Our proposal method could correct for the unexpected decrease of the biological effectiveness caused by interruption time. For photon radiotherapy, the interruption causes the sublethal damage repair. The current study proposed the dose compensation method for the decrease of the biological effect by the interruption.

Changes in the Proliferation Rate, Clonogenicity, and Radiosensitivity of Cultured Cells During and After Continuous Low-Dose-Rate Irradiation.

We investigated the effects of continuous low-dose radiation on proliferation, clonogenicity, radiosensitivity, and repair of DNA double-strand breaks (DSBs) in human salivary gland (HSG) tumor cells. Human salivary gland cells were cultured on acrylic boards above very-low-dose (4.3 μSv/h) or low-dose (27 μSv/h) radiation-emitting sheets or without sheets. Total cell numbers and plating efficiencies were compared among the 3 groups every 1 or 2 weeks until 6 weeks after starting culture. At 2, 4, and 6 weeks, surviving fractions of HSG cells after irradiation at 2 to 8 Gy cultured on the very-low-dose or low-dose sheets were compared to those of the control. At 4 weeks, HSG cells irradiated at 2 Gy were assessed for phosphorylated histone (γH2AX) foci formation, and DSBs were evaluated. No significant differences were observed in total cell number or plating efficiencies with or without low-dose-emitting sheets. The surviving fractions after irradiation of the very-low-dose group at 2 to 6 weeks and those of the low-dose group at 2 to 4 weeks were higher than those of the control (P < .01). Thus, a radioadaptive response was clearly demonstrated. From the γH2AX foci quantification, the adaptive responses were considered to be associated with the efficient repair of DSB, especially slow repair, in this cell line.

Immunoreactivity patterns of tight junction proteins are useful for differential diagnosis of human salivary gland tumors

The expression pattern of tight junction proteins (TJPs) varies among organs and tumor types. In this study, we examined the immunoreactivity of claudin (CLDN)-1, -4, and -7, and JAM-A in salivary gland tumors (SGTs) by histological types and cell types to estimate their usefulness as differential diagnostic markers. Immunoreactivity of CLDN1 was higher in ductal epithelium cells of SGTs than in non-tumor tissues. Conversely, immunoreactivity of CLDN1 was significantly decreased in basal/myoepithelium cells of SGTs compared with that in non-tumor tissues. There was no significant difference between the immunoreactivity of CLDN1 in benign tumors and that in malignant tumors. Immunoreactivity of CLDN4, CLDN7, and JAM-A in ductal epithelium cells was higher in many SGTs than in non-tumor tissues. There was a difference depending on the histological type of SGT in immunoreactivity of CLDN4, CLDN7, and JAM-A in basaloid/myoepithelial cells. It was possible to classify SGTs by a hierarchical clustering using immunoreactivity of TJPs. The results suggest that an immunohistochemical marker panel including these TJPs may be useful for differential diagnosis of SGTs and that CLDN1 is associated with tumorigenesis of SGTs.

Cooperation Between Pten and Smad4 in Murine Salivary Gland Tumor Formation and Progression

Salivary gland tumor (SGT) is a rare tumor type, which exhibits broad-spectrum phenotypic, biological, and clinical heterogeneity. Currently, the molecular mechanisms that cause SGT pathogenesis remain poorly understood. A lack of animal models that faithfully recapitulate the naturally occurring process of human SGTs has hampered research progress on this field. In this report, we developed an inducible keratin 5-driven conditional knockout mouse model to delete gene(s) of interest in murine salivary gland upon local RU486 delivery. We have deleted two major tumor suppressors, Pten, a negative regulator of the PI3K pathway, and Smad4, the central signaling mediator of TGFβ pathway, in the murine salivary gland. Our results have shown that deletion of either Pten or Smad4 in murine salivary gland resulted in pleomorphic adenomas, the most common tumor in human SGT patients. Deletion of both Pten and Smad4 in murine salivary gland developed several malignancies, with salivary adenoid cystic carcinoma (SACC) being the most frequently seen. Molecular characterization showed that SACC exhibited mTOR activation and TGFβ1 overexpression. Examination of human SGT clinical samples revealed that loss of Pten and Smad4 is common in human SACC samples, particularly in the most aggressive solid form, and is correlated with survival of SACC patients, highlighting the human relevance of the murine models. In summary, our results offer significant insight into synergistic role of Pten and Smad4 in SGT, providing a rationale for targeting mTOR and/or TGFβ signaling to control SGT formation and progression.

Adaptation of stochastic microdosimetric kinetic model for charged-particle therapy treatment planning

The microdosimetric kinetic (MK) model underestimates the cell-survival fractions for high linear energy transfer (LET) and high dose irradiations. To address the issue, some researchers previously extended the MK model to the stochastic microdosimetric kinetic (SMK) model. In the SMK model, the radiation induced cell-survival fractions were estimated from the specific energies zd and zn absorbed by a microscopic subnuclear structure domain and a cell nucleus, respectively. By taking the stochastic nature of zn as well as that of zd into account, the SMK model could reproduce the measured cell-survival fractions for radiations with wide LET and dose ranges. However, treatment planning based on the SMK model was unrealistic in clinical practice due to its long computation time and huge memory space required for the computation. In this study, we modified the SMK model to shorten the computation time and to reduce the memory space required for the computation. By using the dose-averaged cell-nucleus specific energy per event in the SMK formalism, the stochastic nature of zn was reflected onto the estimated cell-survival fractions. The accuracy of the modified SMK model was examined through the comparison between the estimated and the measured survival fractions of human salivary gland tumor cells and V79 cells. We then implemented the modified SMK model into the in-house treatment planning software for scanned charged-particle therapy to validate its applicability in clinical practice. As examples, treatment plans of helium-, carbon-, and neon-ion beams were made for an orbital tumor case. The modified SMK model could reproduce the measured cell-survival fractions more accurately compared to the MK model especially for high-LET and high-dose irradiations. In summary, the modified SMK model offers the accuracy and simplicity required in treatment planning of scanned charged-particle therapy for wide LET and dose ranges.

The relative biological effectiveness for carbon, nitrogen, and oxygen ion beams using passive and scanning techniques evaluated with fully 3D silicon microdosimeters.

The aim of this study was to measure the microdosimetric distributions of a carbon pencil beam scanning (PBS) and passive scattering system as well as to evaluate the relative biological effectiveness (RBE) of different ions, namely 12 C, 14 N, and 16 O, using a silicon-on-insulator (SOI) microdosimeter with well-defined 3D-sensitive volumes (SV). Geant4 simulations were performed with the same experimental setup and results were compared to the experimental results for benchmarking. Two different silicon microdosimeters with rectangular parallelepiped and cylindrical shaped SVs, both 10 μm in thickness were used in this study. The microdosimeters were connected to low noise electronics which allowed for the detection of lineal energies as low as 0.15 keV/μm in tissue. The silicon microdosimeters provide extremely high spatial resolution and can be used for in-field and out-of-field measurements in both passive scattering and PBS deliveries. The response of the microdosimeters was studied in 290 MeV/u 12 C, 180 MeV/u 14 N, 400 MeV/u 16 O passive ion beams, and 290 MeV/u 12 C scanning carbon therapy beam at heavy ion medical accelerator in Chiba (HIMAC) and Gunma University Heavy Ion Medical Center (GHMC), Japan, respectively. The microdosimeters were placed at various depths in a water phantom along the central axis of the ion beam, and at the distal part of the Spread Out Bragg Peak (SOBP) in 0.5 mm increments. The RBE values of the pristine Bragg peak (BP) and SOBP were derived using the microdosimetric lineal energy spectra and the modified microdosimetric kinetic model (MKM), using MKM input parameters corresponding to human salivary gland (HSG) tumor cells. Geant4 simulations were performed in order to verify the calculated depth-dose distribution from the treatment planning system (TPS) and to compare the simulated dose-mean lineal energy to the experimental results. For a 180 MeV/u 14 N pristine BP, the dose-mean lineal energy yD¯ obtained with two types of silicon microdosimeters started from approximately 29 keV/μm at the entrance to 92 keV/μm at the BP, with a maximum value in the range of 412 to 438 keV/μm at the distal edge. For 400 MeV/u 16 O ions, the dose-mean lineal energy yD¯ started from about 24 keV/μm at the entrance to 106 keV/μm at the BP, with a maximum value of approximately 381 keV/μm at the distal edge. The maximum derived RBE10 values for 14 N and 16 O ions were found to be 3.10 ± 0.47 and 2.93 ± 0.45, respectively. Silicon microdosimetry measurements using pencilbeam scanning 12 C ions were also compared to the passive scattering beam. These SOI microdosimeters with well-defined three-dimensional (3D) SVs have applicability in characterizing heavy ion radiation fields and measuring lineal energy deposition with sub-millimeter spatial resolution. It has been shown that the dose-mean lineal energy increased significantly at the distal part of the BP and SOBP due to very high LET particles. Good agreement was observed for the experimental and simulation results obtained with silicon microdosimeters in 14 N and 16 O ion beams, confirming the potential application of SOI microdosimeter with 3D SV for quality assurance in charged particle therapy.

Absence of myoepithelial cells correlates with invasion and metastasis of Carcinoma ex pleomorphic adenoma

Myoepithelial cells (MECs) are implicated in the development and progression of human salivary gland tumours. Here, we investigate the potential role for MECs in invasion and metastasis of carcinoma ex pleomorphic adenoma (CXPA). Tumour tissues from 40 CXPA patients diagnosed between 1960 and 2014 were obtained. Patient samples were divided into two groups (non-invasive tumours, n=10; and frankly invasive tumours, n=30). Each group was further divided into two subgroups (metastatic tumours and non-metastatic tumours). Immunohistochemistry for MEC markers (α-SMA, CALPONIN, and p63) was performed, and the number and distribution of MECs was quantified. For non-invasive CXPAs, non-metastatic cases (n=8) displayed a significant enrichment in CALPONIN(+) and α-SMA(+) MECs, but not p63(+) MECs, compared with metastatic cases (n=2). Likewise, for frankly invasive CXPAs (n=30), non-metastatic cases showed a significant enrichment for α-SMA(+), CALPONIN(+), and p63(+) MECs compared with metastatic cases (n=15). We demonstrate that non-invasive CXPAs have the potential for metastasis. Furthermore, the tumour capsule may not be the only barrier preventing invasion and metastasis, as a significant reduction in numbers of myoepithelial cells correlates with invasion and metastasis in CXPA patients.

Amino acid transport system - A substrate predicts the therapeutic effects of particle radiotherapy.

L-[methyl-11C]Methionine (11C-Met) is useful for estimating the therapeutic efficacy of particle radiotherapy at early stages of the treatment. Given the short half-life of 11C, the development of longer-lived 18F- and 123I-labeled probes that afford diagnostic information similar to 11C-Met, are being sought. Tumor uptake of 11C-Met is involved in many cellular functions such as amino acid transport System-L, protein synthesis, and transmethylation. Among these processes, since the energy-dependent intracellular functions involved with 11C-Met are more reflective of the radiotherapeutic effects, we evaluated the activity of the amino acid transport System-A as an another energy-dependent cellular function in order to estimate radiotherapeutic effects. In this study, using a carbon-ion beam as the radiation source, the activity of System-A was evaluated by a specific System-A substrate, alpha-[1-14C]-methyl-aminoisobutyric acid (14C-MeAIB). Cellular growth and the accumulation of 14C-MeAIB or 14C-Met were evaluated over time in vitro in cultured human salivary gland (HSG) tumor cells (3-Gy) or in vivo in murine xenografts of HSG tumors (6- or 25-Gy) before and after irradiation with the carbon-ion beam. Post 3-Gy irradiation, in vitro accumulation of 14C-Met and 14C-MeAIB decreased over a 5-day period. In xenografts of HSG tumors in mice, tumor re-growth was observed in vivo on day-10 after a 6-Gy irradiation dose, but no re-growth was detected after the 25-Gy irradiation dose. Consistent with the growth results, the in vivo tumor accumulation of 14C-MeAIB did not decrease after the 6-Gy irradiation dose, whereas a significant decrease was observed after the 25-Gy irradiation dose. These results indicate that the activity of energy dependent System-A transporter may reflect the therapeutic efficacy of carbon-ion radiotherapy and suggests that longer half-life radionuclide-labeled probes for System-A may also provide widely available probes to evaluate the effects of particle radiotherapy on tumors at early stage of the treatment.

The motogenic effect of EGF and TGF-α on the migration of tumor cells from the oral region

Aim: Epithelial-to-mesenchymal transition (EMT) is recognized as a hallmark of cancer. The change in phenotype of epithelial cells enables them to migrate and the cancer therefore, to become more aggressive. The presence of the epidermal growth factor receptor (EGFR) has been implicated in the spread and aggressive nature of oral tumors. The aims of this present study were to investigate the role of two different growth factors, both ligands of the receptor, and their influence on EMT and cellular motility. Methods: A number of assays were used to investigate the response of the cells to the two growth factors (EGF and transforming growth factor (TGF-α)). These techniques included the 2-D scratch assay, a 2-D-cell scatter assay, and immunocytochemistry. Normal keratinocytes (HaCaT), oral adenoid squamous cell carcinoma (TYS), and human salivary gland tumor cells were used in this study. Results: The results of the scratch and scatter assays indicated that EGF and TGF-α exerted differential effects upon the cells. EGF and TGF-α stimulated the migration of cancer cells in scratch assays. Scatter assays have revealed that both EGF and TGF-α induce EMT. EGF- and TGF-α-induced scattering is EGFR dependent and localization of EMT markers (E-cadherin and vimentin) might play an important role in scattering. These responses were also found to be dependent upon cell type, indicating that the assay could also be used as a simple screen for active growth factor. The observed effects were also dose dependant (data not shown). Conclusion and implication for translation to clinic: We have previously reported that although EGF and TGF-α have ultimately similar effects on motility, it would appear that different mechanisms are responsible. This data extends our knowledge of the action of these growth factors to epithelial cell lines and will inform future testing of the effects of EGF and TGF-α inhibitors.

Human papillomavirus and salivary gland neoplasia: a p16INK4 immunohistochemical and in situ hybridisation study.

This study aimed to evaluate the association between human papillomavirus infection and salivary gland tumours in a Scottish cohort. Specimens from a range of salivary gland tumours operated on between 1997 and 2012 were studied. A tissue microarray constructed from tissue blocks was subjected to p16INK4 (cyclin-dependent kinase inhibitor 2A) immunohistochemistry and in situ hybridisation using probes specific for human papillomavirus, including types 16 and 18. A total of 61 tumours (benign and malignant) were deemed suitable for the study. p16INK4 staining yielded three (4.9 per cent) positive samples: one small cell carcinoma, one squamous cell carcinoma and one poorly differentiated carcinoma. Human papillomavirus in situ hybridisation demonstrated a positive signal in the latter sample only (1.6 per cent). This study demonstrated a very low human papillomavirus detection rate in salivary gland tumours. It can therefore be concluded that human papillomavirus infection is unlikely to play a role in salivary gland neoplasia. Rare human papillomavirus positive cases should be carefully evaluated to exclude the possibility of a metastatic lesion.

Relative clinical effectiveness of carbon ion radiotherapy: theoretical modelling for H&N tumours.

Comparison of the efficiency of photon and carbon ion radiotherapy (RT) administered with the same number of fractions might be of limited clinical interest, since a wide range of fractionation patterns are used clinically today. Due to advanced photon treatment techniques, hypofractionation is becoming increasingly accepted for prostate and lung tumours, whereas patients with head and neck tumours still benefit from hyperfractionated treatments. In general, the number of fractions is considerably lower in carbon ion RT. A clinically relevant comparison would be between fractionation schedules that are optimal within each treatment modality category. In this in silico study, the relative clinical effectiveness (RCE) of carbon ions was investigated for human salivary gland tumours, assuming various radiation sensitivities related to their oxygenation. The results indicate that, for hypoxic tumours in the absence of reoxygenation, the RCE (defined as the ratio of D50 for photons to carbon ions) ranges from 3.5 to 5.7, corresponding to carbon ion treatments given in 36 and 3 fractions, respectively, and 30 fractions for photons. Assuming that interfraction local oxygenation changes take place, results for RCE are lower than that for an oxic tumour if only a few fractions of carbon ions are used. If the carbon ion treatment is given in more than 12 fractions, the RCE is larger for the hypoxic than for the well-oxygenated tumour. In conclusion, this study showed that in silico modelling enables the study of a wide range of factors in the clinical considerations and could be an important step towards individualisation of RT treatments.

High frequency of loss of PTEN expression in human solid salivary adenoid cystic carcinoma and its implication for targeted therapy.

Salivary gland tumor (SGT) is one of the least studied cancers due to its rarity and heterogeneous histological types. Here, we reported that loss of PTEN expression was most frequently found in the poorly differentiated, high grade solid adenoid cystic carcinomas. Loss of PTEN expression correlated with activation of mTOR by increased phosphorylated S6 ribosome protein. We further functionally studied the role of PTEN in a pair of human SACC cell lines, SACC-83 and SACC-LM. Reduced PTEN level was correlated with the metastasis potential. When we knocked down PTEN in the SACC-83 cell line, we observed increased proliferation and enhanced migration/invasion in vitro, and increased tumor size in vivo. We further tested the therapeutical effect by applying a PI3K/mTOR inhibitor NVP-BEZ235 to both SACC cell lines. Decreased cell proliferation, increased apoptosis, as well as reduced cell migration/invasion were observed in both cell lines upon the NVP-BEZ235 treatment. Moreover, the NVP-BEZ235 treatment in a SGT xenograft mouse model significantly reduced primary tumor size and lung metastasis. Taken together, our results demonstrated that PTEN is a potent tumor suppressor in human SGTs, and targeting PI3K/mTOR pathway may be effective in the targeted therapy for human SGT patients with loss of PTEN expression.

RBE and OER within the spread-out Bragg peak for proton beam therapy: in vitro study at the Proton Medical Research Center at the University of Tsukuba.

There are few reports on the biological homogeneity within the spread-out Bragg peak (SOBP) of proton beams. Therefore, to evaluate the relative biological effectiveness (RBE) and the oxygen enhancement ratio (OER), human salivary gland tumor (HSG) cells were irradiated at the plateau position (position A) and three different positions within a 6-cm-wide SOBP (position B, 26 mm proximal to the middle; position C, middle; position D, 26 mm distal to the middle) using 155-MeV/n proton beams under both normoxic and hypoxic conditions at the Proton Medical Research Center, University of Tsukuba, Japan. The RBE to the plateau region (RBEplateau) and the OER value were calculated from the doses corresponding to 10% survival data. Under the normoxic condition, the RBEplateau was 1.00, 0.99 and 1.09 for positions B, C and D, respectively. Under the hypoxic condition, the RBEplateau was 1.10, 1.06 and 1.12 for positions B, C and D, respectively. The OER was 2.84, 2.60, 2.63 and 2.76 for positions A, B, C and D, respectively. There were no significant differences in either the RBEplateau or the OER between these three positions within the SOBP. In conclusion, biological homogeneity need not necessarily be taken into account for treatment planning for proton beam therapy at the University of Tsukuba.